Method for preparing assay supports having an ordered set of reaction zones

ABSTRACT

The abstract discloses a method for preparing supports for the assay of chemical or biological substances, consisting in preparing N different supports, each being homogeneous for a type of reaction, then in mutually combining, according to a predetermined arrangement, all or part of the said homogenous N supports to constitute assay supports having each an ordered set of reaction zones of N different types or less. The invention also concerns the assay supports prepared by this method and their use in processes of immunological hybridisation assays.

BACKGROUND OF THE INVENTION

The present invention concerns the area of assaying chemical orbiological substances, such as sequences of nucleic acids or proteins byspecific reaction with a set of specific reagents fixed on a support. Itrelates more particularly to the assay of nucleic acids by hybridisationwith oligonucleotide probes fixed on a support. This type of assay findsapplication in sequencing, diagnosis or quality control for research orindustrial production.

The match law A-T (or A-U) and G-C gives nucleic acids the property offorming specific hybridisation complexes between complementarysequences. This long known hybridisation property means that a fragmentof nucleic acid, or an oligonucleotide, can be used to display thepresence of a complementary nucleic sequence. The analysis of DNAfragments after gel separation (E. Southern, J. mol. Biol., 1975, 98,503-517) is today largely used both in the area of fundamental researchand in medical analysis (Caskey, Science, 1987, 236, 1223-1228;Landegrer et al., Science, 1988, 242, 229-237; Arnheim et al., Ann rev.Biochem., 1992, 61, 131-156).

Several assay techniques of nucleic acids based on their hybridisationproperty with probes have been developed, in particular for thesequencing of unknown DNAs or RNAs, the detection of sequencesassociated with a pathology, and the search for point mutations insequences (S. Ikata et al., Nuclei Acids Res., 1987, 15, 797-811; J. A.Matthews, L. J. Krieka, Analytical Biochemistry, 1988, 169, 1-25).

More recently, new assay methods for unknown DNA fragments have been putforward, based on hybridisation with a series of oligonucleotidescomprising 5 to 9 bases arranged in known sequence and immobilised on asolid support (E. Southern, European Patent published under number: 0373 203; K. R. Khrapko et al., FEBS Letters, 1989, 256, 118-122 ; R.Drmanac et al., Genomics, 1989, 4, 114-128 ; R. Drmanac et al., DNA andCell Biology, 1990, 9, 527-534 ; K. R. Khrapko et al., J. DNA SequencingMapp., 1991, 1, 375-388 ; R. Drmanac et al., Science 1993, 260,1649-1652 ; R. J. Lipshutz, J. Biomol. Struct. Dyn., 1993, 11, 637-653;U. Maskos, E. Southern, Nucleic Acids Res., 1993, 21, 4663-4669 ; A. C.Pease et al., Proc. Natl. Acad. Sci., USA, 1994, 91, 5022-5026; J. C.Williams, Nucleic Acids Res., 1994, 22, 1365-1367 ; E. M. Southern,Nucleic Acids Res., 1994, 22, 1368-1373.

These new assay methods are based on the preparation of a support onwhose surface is fixed a series of oligonucleotide sequences having thesame length covering all possible sequences for this length, each of theoligonucleotide sequences occupying cells, or zones, separated on thesupport. The preparation and use of said support is described inparticular in European patent applications published under N°s 373 203and 392 546 and in international patent application published under n°WO 94/12670. The sequence of nucleic acid to be assayed is labelled andapplied to the support under conditions allowing hybridisation with theoligonucleotides, then after washing, the localisation of the labellingis noted on the support surface in order to detect the signalstransmitted by any hybrids which may be formed between the assayed,labelled DNA fragment and one or more oligonucleotides of the seriesimmobilised on the solid support. Each position shown to be positive onthe support corresponds to a complementary sequence thus identified inthe assayed DNA fragment.

Immobilisation of the oligonucloetides on the support surface may beconducted in accordance with two principles: either immobilisation aftersynthesis of the oligonucleotides, or direct synthesis of theoligonucleotides on the support.

Immobilisation after synthesis of the oligonucleotides is difficult toimplement and does not appear sufficiently reproducible as is the casefor antibodies ( European patent N° 0 063 810), therefore most teamsworking on this subject have chosen to develop techniques for directsynthesis on the support to be used for hybridisation and detection(International Patent Applications WO 90 03 382, WO 90 15 070, WO 91 08307, WO 91 07 087, WO 92 10 092, WO 92 10 587, WO 93 10 161, WO 93 09668, WO 94 22 889, WO 95 11 748, WO 95 30 774). These involve thesynthesis one same surface of a series of oligonucleotide sequenceshaving the same length covering all possible sequences for this length.Therefore, for oligonucleotides with 2 bases, chosen from among the fourforming the DNA, 16 sequences are possible, for oligonucleotides with 3bases 64 sequences are possible, for oligonucleotides with 4 bases 256sequences are possible, for oligonucleotides with 5 bases 1024 sequencesare possible, for oligonucleotides with 6 bases 4096 sequences arepossible etc.. The shorter the oligonucleotide sequence, the lessinformation it provides for sequencing; for a DNA fragment witharrangement, all or part of said homogeneous supports are mutuallycombined to form assay supports each having an ordered set of reactionzones of N different types or less.

DETAILED DESCRIPTION OF THE INVENTION

By “type of reaction ” is meant the specific reaction likely to occurbetween the assayed substance and the support, when they are broughtinto contact. This reaction is determined by the nature of the differentN reagents, present in ordered and verifiable manner on the supportsobtained by the method of the invention, each of the initial N supportsbeing homogeneous for one of its reagents. For the assay of nucleicacids, by type of reaction is meant the specific hybridisation reactionlikely to occur between an oligonucleotide sequence fixed on one of theinitial N supports, re-detected in a reaction zone on the final support,and a complementary nucleic acid sequence present in the assayedsubstance.

According to one first particular embodiment, the method of theinvention comprises the following stages:

a) preparation of N flat shaped supports homogeneous for a type ofreaction;

b) cutting said flat, homogeneous N supports into strips ofsubstantially equal width;

c) combining substantially side by side the strips derived from at leastone part of the N homogeneous supports so as to prepare intermediatesupports which, along an axis that is perpendicular to said strips ofhomogeneous supports, comprise alignments of different reaction zones,each of said alignments of one same intermediate support beingidentical;

d) cutting the intermediate supports into strips of substantially thesame width, each of said strips comprising one of said alignments ofdifferent reaction zones,

e) combining substantially side by side the strips which each comprisean alignment of different reaction zones, in such manner as to formassay supports each having alignments of ordered reaction zones of Ndifferent types or less.

According to a second particular embodiment, the method of the inventioncomprises the following stages:

a′) preparation of N homogeneous supports in the form of filaments orfibres, and

b′) combining substantially side by side several of said filaments insuch manner as to prepare intermediate supports which, along an axisperpendicular to said filaments, comprise alignments of differentreaction zones, each of said alignments of one same intermediate supportbeing identical, then, carrying out stages (d) and (e) previouslydescribed in the first embodiment.

Stage (b′) above may consist of gluing side by side the homogeneousfilaments prepared in stage (a′) so as to form the intermediate supportsdefined in stage (c) of the first embodiment.

One variant of stages (a) and (a′) described above, consists of:

a″) preparing N homogeneous supports in the form of filaments, andproceeding with weaving each of said N filaments with a weft made ofreactively neutral strand so as to prepare the N homogeneous supports ofthe type of those in stage (a) described in the first embodiment,

then, carrying out stages (b), (c), (d) and (e) previously described.

One variant of stages (a″) and (b′) described above consists of:

a′) preparing N homogeneous supports in the form of filaments, and

b″) proceeding with weaving all or part of the homogeneous N filamentsby intertwining a weft made of said N filaments with a strand that isreactively neutral, so as to prepare intermediate supports or a finalsupport, then

optionally carrying out stages (d) and (e) described previously, if thesupports prepared at stage (b″) are intermediate supports.

In order to maintain the rigidity of the intermediate support of stages(b), (b′)and (b″), it is advantageously fixed to an auxiliary elementfor temporary or permanent support; “temporary” meaning that theauxiliary support element is removed after stage (e) or (b″),“permanent” meaning that the auxiliary support element is left in placeafter stage (e) or (b″).

Also, the final support obtained at stages (e) and (b″) mayadvantageously be made rigid and therefore handled with greater ease ifit is fixed onto a support element. Also, this support element may beused to give a particular shape to the final support, such as forexample one or more recesses to facilitate and border the deposits ofhybridisation and revealing reagents at each reaction zone, or even toform an edge for the support.

The homogeneous supports of flat shape or in fibre form may be in aporous or non-porous matter. As porous flat support, preference is givento a cellulose microporous membrane.

If the support of the invention is intended to be used for the assay ofnucleic acids, the initial N supports are each homogeneous for one typeof specific hybridisation reaction. Advantageously, each of the Nhomogeneous supports then comprises a different oligonucleotidesequence. In one particular embodiment, intended for the sequencing orassay of mutations, on each of the N homogeneous supports is fixed adifferent oligonucleotide sequence but of similar length, all theoligonucleotide sequences of the N supports then covering all thepossible sequences for this length.

Assay of the extent of hybridisation with each of the oligonucleotides,for example using the Tm values, at each reaction zone of the supports,can determine with great precision the sequence of the assayed nucleicacid, DNA or RNA or the presence of a mutation.

The supports of the inventions may also be used for immunological assay;in this case, the N supports are each homogeneous for one type ofantigen-antibody reaction. In this embodiment, one different antibody orone antigen, according to whether the assayed substance is an antigen oran antibody, is fixed by any method known to men of the art on each ofthe initial N supports.

The supports of the invention may also be used for the assay ofsubstrates likely to react with a series of different enzymes eachplaced at the level of each reaction zone.

The invention also relates to an assay support prepared using theabove-described methods, and the use of this support for methods ofassaying chemical or biological substances, and its incorporation intoassay kits for chemical or biological substances.

Other characteristics and advantages of the invention will becomeapparent on reading the following examples which are given for guidancepurposes and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

One example of the method for preparing an assay support of theinvention comprises stages (a) to (e) described below with reference toFIGS. 1 to 7 of the appended drawings.

a) N homogeneous supports are prepared for one type of reaction, forexample, by fixing a different oligonucleotide sequence on each of the Nsupports.

For a clearer understanding of the method of the invention, FIGS. 1, 2and 3 only show three supports of a series of N homogeneous supports offlat shape:

the support in FIG. 1 is homogeneous for the type of reaction referenced(1) corresponding to a first nucleic sequence;

the support in FIG. 2 is homogeneous for the type of reaction referenced(2) corresponding to a second nucleic sequence;

the support in FIG. 3 is homogeneous for the type of reaction referenced(N) corresponding to a nth nucleic sequence.

b) In each of the N homogeneous supports, strips of substantially thesame width are cut as shown by the vertical lines in FIGS. 1, 2 and 3;therefore each of the homogeneous supports of FIGS. 1 to 3 is cut into 9strips.

c) Using these strips, the intermediate supports of FIGS. 4, 5 and 6were prepared by placing strips derived from stage (b) side by side, onan auxiliary support element, It can be easily seen that, along thehorizontal axes perpendicular to the strips derived from the homogeneoussupports, each of the intermediate supports comprises alignments ofdifferent reaction zones, each of said alignments of one sameintermediate support being identical. FIGS. 4, 5 and 6 show three ofthese intermediate supports, each made up of 8 strips prepared at stage(b)

The support in FIG. 4 comprises 8 different vertical strips eachcarrying the same type of reaction zone, and 15 identical horizontalstrips each comprising an alignment of 8 reaction zones of differenttypes referenced: 1, 2, 3, 4, 5, 6, 7 and 8.

The support in FIG. 5 comprises an identical layout to that in FIG. 4,but with an alignment of 8 reaction zones of different type to those ofthe support in FIG. 4 referenced: 9, 10, 11, 12, 13, 14, 15 and 16.

the support in FIG. 6 represents the last intermediate support of theseries of intermediate supports, on which the strips are laid out as inFIGS. 4 and 5 with an alignment of 8 different reaction zonesreferenced: N-7, N-6, N-5, N-4, N-3, N-2, N-1 and N.

d) the intermediate supports prepared at stage (c) were cut into stripsof substantially the same width, each of said strips comprising one ofsaid alignments of different reaction zones. In this way, from eachintermediate support, a set of identical strips is obtained, each setbeing different. The set of strips derived from the intermediate supportin FIG. 4 comprises 15 strips of different zones referenced: 1, 2, 3, 4,5, 6, 7 and 8; the set of strips derived from the intermediate supportin FIG. 5 comprises 15 strips of different zones referenced: 9, 10, 11,12, 13, 14, 15 and 16, the set of strips derived from the intermediatesupport in FIG. 6 comprises 15 strips of different zones referenced:N-7, N-6, N-5, N-4, N-3, N-2, N-1 and N.

e) the assay supports are prepared by placing side by side, on apermanent support element, a strip of each of the sets of identicalstrips prepared at stage (d) in such manner that each of the assaysupports contains the N different reaction zones. The example of FIG. 7,shows one of the 15 assay supports which may be obtained by using allthe strips of stage (d).

I—Preparation of the N Homogeneous Supports of Stage (a) 1) SupportStructure

The homogeneous supports of stage (a) may be porous or non-porous, andof flat or fibrous shape. By way of example, the supports of table Ibelow can be cited.

TABLE I SUPPORTS Flat Fibrous Non-porous sheets, films strands, meshesPorous paper, non-woven sheeting, micro mono-filaments, andultra-filtration membranes strands, multi- filaments, hollow fibres formicro and ultrafiltration.

The choice of a porous or non-porous support is dependent upon thedensity of the reagents it is desired to deposit at each reaction zone.Owing to the greater available surface area, porous supports offer thepossibility of achieving high molecule density.

The preferred porous supports of flat shape are microfiltrationmembranes with a pore size of between 0.1 and 10 μm and a thickness ofbetween 50 and 150 μm. Strands are also a porous support of interest asthey combine large surface area with high mechanical stability and theirthickness is between 50 and 500 μm.

2) The Matter of Which the Support is Made

The matter of which the homogenous support is made having either aporous or non-porous structure, of fibrous or flat shape as describedabove, is generally an organic polymer, but in some cases, such assheets or films, it may be a metal, aluminum for example. If the reagentelements are fixed chemically by covalent bonds with the support, anypolymer allowing this type of bonding may be used. Among the latter,mention may be made of cellulose, polyamides, polyurethanes,polystyrenes and polyacrylonitryls or their derivatives. In the case ofmicrofiltration membranes or fibres, preference is given to cellulosehowever as this material combines good chemical reactivity with strongstability against heat and organic solvents, high hydrophylicity and lownon-specific absorption. The hydrophylic property is of importance forwetting with aqueous samples. The cellulose used may be in natural orfibrous form, for example of cotton filament type, but may also be ofregenerated product type, such as viscose fibres. Microporous cellulosemembranes, in all cases, contain regenerated cellulose, obtained bysaponification of cellulose acetate membranes.

Conversely, strong absorption of the support is prejudicial if thereagent elements are fixed on the support by absorption. Antigens andantibodies are easily absorbed on cellulose nitrate. Polyamides are alsoof interest if absorption is used to fix the reagent elements on thesupport, also hydrophobic polymers such as polyolefins (polyethylene andpolypropylene), polysulphones and polyethersulphones.

3—Type of Reaction

The reagents which may react with the assayed substance at each reactionzone are fixed on the supports using known chemical or physical methodsof the prior art. They may be antigen proteins able to react with anantibody, or conversely antibodies able to react with a protein or partof a protein. They may also, and it is one of the main applications ofthe invention, be probe oligonucleotides, which are fixed on thepreviously described support using any method known to men of the art.Immobilisation may be made after synthesis of the oligonucleotide, ordirect synthesis of the oligonucleotides on the support may be madeusing covalent bonds or a fixation arm which may assume the priorpreparation of the N supports. Physical fixation may also be conducted,by absorption, ion exchange, chelate formation or any other equivalenttechnique known to men of the art.

II—Preparation of the Strips and Intermediate Supports of Stages (b)and 1) The Strips

If the support prepared at stage (a) is flat, it is cut directly intostrips. For fibrous supports, these are advantageously fixed on asupport element. If N is a number of great size, it is preferable thatstrip width should be as narrow as possible in order to achieve assaysupports of small size. Therefore, a strip width of approximately 0.1 mmseems reasonable taking into account the cutting means available. Forother applications, in which the size of the assay support is notcritical, it is possible to use strips with an approximate width of 5mm.

2) Intermediate Supports

The strips or fibres are placed on the auxiliary support elements,either side by side, or directly in contact with one another, or leavinga small space in the region of 0.1 to 1 mm between two strips in orderto facilitate accurate positioning of each strip.

The auxiliary support element is a sheet or flat film in any type ofmaterial able to stabilize the strips forming the intermediate supportand the final support.

The use of these auxiliary support elements may be solely temporary, thefinal assay support being removed from the auxiliary support element atthe end of the process, or it may be permanent, the auxiliary supportelement then forming an integral part of the assay support of theinvention. It is generally preferred to make temporary use of theseauxiliary support elements.

In the event of permanent use, the fixation of the strips on theauxiliary support elements must be stable in order not to interfere withassay conditions when the support is used, for example, withhybridisation conditions if the reagent elements of the reaction sitesare oligonucleotides.

Permanent fixation of the strips on the auxiliary support element may bemade by various means; it may be achieved by heat lamination forexample, welding, gluing. For strips prepared from homogeneous supportsmade of microporous cellulose membranes, they may be fixed in permanentmanner on a polyolefin sheet (polypropylene, polyethylene or a mixturethereof) by heat lamination. In this case, the sheet is heated underpressure to the region of its melting point so that the melted productpenetrates the membrane pores and creates a homogeneous mechanical bondbetween the support and the auxiliary support element after cooling.Another possibility consists of using reactive adhesives and resins,especially containing epoxides, urethanes or acrylates. Generally,non-reactive adhesives do not offer sufficient adhesive stability in thewet state.

Temporary fixation of the strips on the auxiliary support element may bemade by heat lamination at temperatures and/or pressures that areinsufficient to produce permanent fixation, or using adhesives havinglow adhesivity. As an example, an adhesive cartridge can be used of thetype marketed by Beiersdorf (Hamburg, Germany) under the name“Tesafilm”.

III—Cutting the Intermediate Supports of Stage (d)

The intermediate supports prepared in the previous stage are made up ofstrips derived from several initial homogeneous supports fixed on theauxiliary support element. These supports must be cut with greatprecision and the cutting stage is therefore a critical phase. Theinitial homogeneous support strips and the auxiliary support elementmust not be damaged ; also the position of the homogeneous supportstrips on the auxiliary element must not be changed when cutting. Thelower limit of the width of the strips cut in the intermediate supportdepends upon the means available for cutting and holding the support.The standard most sophisticated means such as Laser can achieve a widthof approximately 0.1 mm, but generally a strip width greater than thatof the strips prepared in stage (b) is tolerated. If, for example, astrand having a thickness of 50 μm is used to prepare the homogeneoussupport, a cutting width at stage (d) in the region of 1 to 2 mm canallow a great number of reagent molecules to be fixed at a reactionzone. However, for certain applications in which the number of reagentmolecules is not critical, the width of the strips prepared at stages(b) and (d) may be in the region of several millimeters.

IV—Preparation of the Assay Supports of Stage (e)

The assay supports must be geometrically stable at every stage ofapplication. They must not bulge or shorten, for example when they aresubmitted to hybridisation conditions, they must remain sufficientlyrigid so that they can be easily handled. Polymer films or sheets havethese properties; among the latter mention may be made of the followingexamples: a film of polyethylene-glycolterephtalate, having a thicknessof 50 to 500 μm, advantageously from 100 to 250 μm, or polyolefin sheetshaving similar thickness.

If at stage (c) the homogeneous support strips of stage (a) were fixedon auxiliary support elements in permanent manner, the assay supportsare prepared by fixing the strips prepared at stage (d) side by side ona permanent support element; the side of the strips which is fixed onthe permanent auxiliary support element is the side corresponding to theauxiliary support element.

If at stage (c) the intermediate support strips were fixed in temporarymanner on the auxiliary support element, the assay supports are preparedby fixing the strips prepared at stage (d) side by side on a permanentsupport element ; the side of the strips which is fixed on the permanentsupport element is the side corresponding to the homogeneous supportstrips, the auxiliary support element being removed. Final fixation ofthe strips on the permanent support element is conducted as described instage (c) for permanent fixation of the strips on the auxiliary supportelements.

V—DETAILED EXAMPLES 1) Example n° 1

a) N microporous membranes of regenerated type SM 18606 marketed bySartorius AG (Göttingen, Germany) are used having a pore size of 0.45μm. On each of these N membranes, direct synthesis is made or a type ofoligonucleotide sequence is fixed so as to obtain N membranes each beinghomogeneous for one type of the N oligonucleotide sequencespre-determined in relation to the sequences of nucleic acids it isdesired to assay.

b) Synthesis of the N oligonucleotides of pre-determined sequences maybe made with an automatic synthesizer using the phosphoramidite method,either directly on the membranes determined above under (a), or onanother synthesis support, and in this case by detaching theoligonucleotides from the synthesis support during the last deprotectionstage, then by fixing the oligonucleotides by any appropriate functionon the cellulose membranes determined under (a).

c) Strips having a width of 2 mm are cut in each of the N homogeneoussupports previously prepared, then the different strips are placed sideby side on sheets of a polypropylene and polyethylene mixture of FO 2432type, FA Freudenberg, and submitted to lamination at a temperature of130-135° C. under a pressure of 10N/cm² and a speed of 5 m/min (lengthheated: 1.5 m).

The intermediate supports prepared in this way are cut in turn intostrips 2 mm wide perpendicular to the cutting direction of the initialhomogeneous N supports, then deposited with their sheet on a secondsheet, laminated a second time under the same conditions as previously.

2) Example n° 2

Identical to example 1, but at the last stage the strips are glued withan acrylate adhesive of Macbond B2112 type, Fa. Mantac on one side ontoa polymer film, and on the other side onto a polyester film of BN 180type, Fa. Kalle, Wiesbaden.

3) Example n° 3

N homogeneous cotton filaments 200 μm thick each carrying a differentoligonucleotide sequence are prepared as in example 1 for a cellulosemembrane.

Different filaments are glued side by side on adhesive cartridges of“Scotch Klebeband ablösbar” type made by 3 M (USA) which are cut intostrips 5 mm wide in a direction perpendicular to the filament. Thestrips prepared in this way are glued onto a Macbond B 212 cartridge onthe filament side, whereas the other side of the Macbond cartridge isglued to a polyester film as in example 2, and the Scotch cartridgestrips are removed to provide access to the filament pieces.

What is claimed is:
 1. A method for preparing supports for assaying achemical or biological substance, said supports each having an orderedset of reaction zones, the method comprising: (a) preparing a set ofhomogeneous strips for each reaction zone, (b) grouping several of saidstrips from step (a) substantially edge to edge so as to prepareintermediate supports which, along an axis perpendicular to said strips,comprise alignments of different reaction zones, and which, along asecond axis comprise alignments of identical reaction zones, (c) cuttingeach intermediate support into a second set of strips, havingsubstantially the same width, each of said strips from the second set ofstrips comprising one of said alignments of different reaction zones,and (d) grouping the strips from step (c) each comprising an alignmentof different reaction zones substantially edge to edge so as to formassay supports each having alignments of ordered reaction zones ofdifferent types.
 2. A method according to claim 1, wherein theintermediate support is fixed onto a temporary or permanent auxiliarysupport element.
 3. A method according to claim 1, wherein the assaysupport is fixed on a support element.
 4. A method for preparingsupports for immunological assays according to claim 1, wherein thehomogeneous strips are homogeneous for one type of antigen-antibodyreaction.
 5. A support for the assay of chemical or biologicalsubstances prepared by a method according to claim
 1. 6. A method forthe assay of chemical or biological substances, wherein the method isconducted with at least one support defined in claim
 1. 7. An assay kitfor chemical or biological substances, wherein the kit comprises atleast one assay support defined in claim
 1. 8. A method according toclaim 1, wherein the homogeneous strips are in a porous or non-porousmatter.
 9. A method according to claim 8, wherein the homogeneous stripsare cellulose membranes.
 10. A method for preparing supports for theassay of nucleic acids in accordance with claim 1, wherein thehomogeneous strips are homogeneous for a type of specific hybridizationreaction.
 11. A method according to claim 1, wherein a differentoligonucleotide sequence is fixed on each set of homogeneous strips. 12.A method according to claim 11, wherein a different oligonucleotidesequence of equal length is fixed on each set of homogeneous strips, allthe oligonucleotide sequences of each set of homogeneous strips coveringall the possible sequences for this length.
 13. A method according toclaim 1, wherein the homogeneous strips are prepared from a homogeneoussupport of flat shape.
 14. A method according to claim 13, wherein thehomogeneous strips are prepared by cutting the homogeneous support intostrips of substantially the same width.
 15. A method according to claim1, wherein the homogeneous strips are in filament form.
 16. A methodaccording to claim 15, wherein the homogeneous strips in filament formare woven with a strand made of reactively neutral thread to prepare ahomogeneous support of flat shape.